Molecular imaging of T4 phage in mammalian tissues and cells - PubMed (original) (raw)

Molecular imaging of T4 phage in mammalian tissues and cells

Zuzanna Kaźmierczak et al. Bacteriophage. 2014.

Abstract

Advances in phage therapy encourage scientific interest in interactions of phages with human and animal organisms. This has created a need for developing tools that facilitate studies of phage circulation and deposition in tissues and cells. Here we propose a new green fluorescent protein (GFP)-based method for T4 phage molecular imaging in living systems. The method employs decoration of a phage capsid with GFP fused to the N-terminus of Hoc protein by in vivo phage display. Fluorescent phages were positively assessed as regards their applicability for detection inside living mammalian cells (by phagocytosis) and tissues (filtering and retention by lymph nodes and spleen). Molecular imaging provides innovative techniques that have brought substantial progress in life sciences. We propose it as a useful tool for studies of phage biology.

Keywords: T4 phage; green fluorescence protein; molecular imaging; phage circulation; phagocytosis.

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Figures

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Figure 1. Expression of recombinant GFP-Hoc fusion in E. coli. Expression plasmids containing GFP-hoc fusion were tested for their effectiveness in production of GFP-Hoc proteins as showed by SDS-PAGE. M, marker; 1, induced GFP-Hoc expressing bacteria.

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Figure 2. Fluorescence of GFP-Hoc fusion in E. coli. Effective fluorescence of the GFP-Hoc fusion was assessed by comparison of fluorescence in E. coli induced for GFP-Hoc production and control E. coli (expressing protein Hoc without GFP).

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Figure 3. Separation of modified bacteriophages by fast protein liquid chromatography. (A) Fraction containing bacteriophages. (B) Fraction containing non-incorporated GFP-Hoc fusions and other proteins.

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Figure 4. Comparison of fluorescence in GFP-modified and control phage. Effective fluorescence of the purified GFP.Hoc.HAP1 phages was compared with the fluorescence of HAP1 phage (without GFP, purified identically by FPLC).

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Figure 5. Visualization of GFP-modified bacteriophages in phagocytic cells J774A.1 (murine macrophages). Fluorescence of murine macrophage cell line was visualized in fluorescent microscopy. (A) control, phagocytes before incubation with GFP.Hoc.HAP1 phage, (B) phagocytes after 40 min incubation with GFP.Hoc.HAP1 phage.

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Figure 6. Visualization of GFP-modified bacteriophages in murine tissues. GFP.Hoc.HAP1 phages and non-modified HAP1 phages (control) were injected into murine lateral tail vein (1011 pfu/mouse). Fluorescence of spleen and lymph nodes was visualized in an imaging hood. (A) spleen of mice injected with non-modified HAP1 phage, (B) spleen of mice injected with GFP.Hoc.HAP1 phage, (C) lymph node of mice injected with non-modified HAP1 phage, (D) lymph node of mice injected with GFP.Hoc.HAP1 phage.

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